FIGURE 7.

Immortalized BRCA1- or BRCA2-mutant cells display elevated NFκB activity. A, PIK3CA^H1047R distinctly influences differentiation properties of immortalized BRCA1 and BRCA2 mutation carriers. Vector control or PIK3CA^H1047R-expressing breast epithelial cell lines from non-carriers (KTB34 and KTB39) and BRCA1 or BRCA2 mutation carriers were stained with indicated antibodies and characterized by flow cytometry (n = 3). Representative data are shown. PIK3CA^H1047R robustly increased EpCAM expression and increased differentiated phenotype of BRCA1-mutant cells. CD49f⁺/EpCAM⁺ cells increased from 12% to 36% whereas CD49f⁺/EpCAM⁻ cells decreased from 80% to 34% upon PIK3CA^H1047R overexpression. B, BRCA1 mutant cells display elevated phosphorylation of p65, a NFκB subunit, which indirectly suggests activation of NFκB. Expression levels of PIK3CA in cells transduced with PIK3CA-mutant virus are shown. Regular growth media condition had to be used to detect PIK3CA-mutant overexpression because of robust induction of endogenous PIK3CA upon serum starvation. An antibody that preferentially recognizes PIK3CA^H1047R mutant was used in the Western blot analysis. The same extract was used to measure pAKT(S473) and AKT to ensure that PIK3CA^H1047R is functional in transduced cells. C, Approximately 30% of breast cancers in BRCA1 or BRCA2 mutation carriers carry PIK3CA mutations. Data were generated using cBioportal (54). D, Immortalized BRCA1 or BRCA2 mutant cells but not cells from non-carriers are sensitive to DMAPT. PIK3CA^H1047R-overexpressing cells, irrespective of BRCA mutation status, were sensitive to DMAPT (statistical test used—one-way ANOVA). E, Immortalized cell lines from BRCA1 or BRCA2 mutation carriers express higher levels of MIR205HG (statistical test used—unpaired t test). Note that PIK3CA^H1047R, which robustly induced differentiation of BRCA1-mutant cells, reduced MIR205HG levels in these cells. P values, *<0.05; **<0.01. Immortalized BRCA1/2-mutant cells express BRCA1 and BRCA2 transcripts at variable levels with BRCA2-mutant cells expressing significantly higher levels of BRCA1 transcripts compared with non-carrier cells (statistical test used—one-way ANOVA).